Human CCN-like growth factor

ABSTRACT

A human Small CCN-Like Growth Factor polypeptide (SCGF) and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for wound healing or tissue regeneration, stimulating implant fixation and angiogenesis. Antagonist against such polypeptides and their use as a therapeutic to treat atherosclerosis, tumors and scarring are also disclosed. Diagnostic assays for identifying mutations in SCGF nucleic acid sequences and altered levels of the SCGF polypeptide are also disclosed.

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention has been putatively identified as a human smallCCN-like Growth Factor, sometimes hereinafter referred to as “SCGF”. Theinvention also relates to inhibiting the action of such polypeptides.

[0002] The polypeptide of the present invention is related to a familyof growth regulators comprising cef 10/cyr 61, connective tissue growthfactor (CTGF), and nov. The mRNA corresponding to the polypeptide ofthis invention is highly expressed in the kidney, lung, heart and brain,with the abundance in that order.

[0003] Growth factors and other mitogens, including transformingoncogenes, are capable of rapidly inducing a complex set of genes to beexpressed by certain cells (Lau, L. F. and Nathans, D., MolecularAspects of Cellular Regulation, 6:165-202 (1991). These genes, whichhave been named immediate early or early response genes, aretranscriptionally activated within minutes after contact with a growthfactor or mitogen, independent of de novo protein synthesis. A group ofthese immediate early genes encodes secreted, extracellular proteinswhich are needed for coordination of complex biological processes suchas differentiation and proliferation, regeneration and wound healing(Ryseck, R.P. et al, Cell Growth Differ., 2:235-233 (1991).

[0004] Highly related proteins which belong to this group include cef 10from chicken, which was detected after induction by the viral oncogenepp6O^(v-arc) (Simmons, D. L. et al., PNAS, U.S.A., 86:1178-1182 (1989).A closely related protein, cyr 61, is rapidly activated by serum orplatelet-derived growth factor (PDGF) (O'Brien, T. P. et al., Mol. CellBiol., 10:3569-3577 (1990). The overall amino acid identity between cef10 and cyr 61 is as high as 83%. A third member is human connectivetissue growth factor (CTGF) (Bradham, D. M. et al., J. Cell. Biol.,114:1285-1294 (1991). CTGF is a cysteine-rich peptide which is secretedby human vascular endothelial cells in high levels after activation withtransforming growth factor beta (TGF-β). CTGF exhibits PDGF-likebiological and immunological activities and competes with PDGF for aparticular cell surface receptor.

[0005] A fourth member of the immediate-early proteins is fisp-12, whichhas been shown to be induced by serum and has been mapped to a region ofthe reurine genome (Ryseck, R. P. et al., Cell Growth Differ., 2:235-233(1991). Yet another member of this family is the chicken gene, nov,normally arrested in adult kidney cells, which was found to beoverexpressed in myeloblastosis-associated virus type 1 inducednephroblastomas. Further, expression of an amino-terminal-truncated novproduct in chicken embryo fibroblasts was sufficient to inducetransformation (Joliot, V. et al., Mol. Cell. Biol., 12:10-21 (1992).

[0006] The expression of these immediate early genes act as “thirdmessengers” in the cascade of events triggered by growth factors. It isalso thought that they are needed to integrate and coordinate complexbiological processes, such as differentiation and wound healing in whichcell proliferation is a common event.

[0007] This emerging family of growth regulators is called the CCNfamily for CTGF; cef 10/cyr 61; and nov. The polypeptide of the presentinvention is thought to be a member of this family of growth regulators.

[0008] In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof.

[0009] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the polypeptide ofthe present invention including mRNAs, DNAS, cDNAs, genomic DNAs as wellas analogs and biologically active and diagnostically or therapeuticallyuseful fragments and derivatives thereof.

[0010] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptide byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a nucleic acid sequenceencoding a polypeptide of the present invention, under conditionspromoting expression of said protein and subsequent recovery of saidprotein.

[0011] In accordance with yet a further aspect of the present invention,there is provided a process of utilizing such polypeptide, orpolynucleotide encoding such polypeptide for therapeutic purposes, forexample, to treat muscle wasting diseases, osteoporosis, to aid inimplant fixation, to stimulate wound healing and tissue regeneration, topromote angiogenesis and to stimulate proliferation vascular smoothmuscle and endothelial cell production.

[0012] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0013] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, to limit theproduction of excess connective tissue during wound healing or pulmonaryfibrosis.

[0014] In accordance with yet a further aspect of the present invention,there are also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to thepolynucleotide sequences of the present invention.

[0015] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting diseases related tothe under-expression and over-expression of the polypeptide of thepresent invention and mutations in the nucleic acid sequences encodingsuch polypeptide.

[0016] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0017] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0018] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0019]FIG. 1 shows the cDNA and corresponding deduced amino acidsequence of the polypeptide of the present invention. The initial 23amino acids reprebent the putative leader sequence (underlined). Thestandard one letter abbreviations for amino acids are used. Sequencingwas performed using a 373 Automated DNA sequencer (Applied Biosystems,Inc.).

[0020]FIG. 2 shows the amino acid sequence homology between thepolypeptide of the present invention and other proteins which aremembers of the CCN family.

[0021] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature polypeptide having the deduced amino acid sequence of FIG. 1 (SEQID NO: 2) or for the mature polypeptide encoded by the cDNA of the clone(s) deposited as ATCC Deposit No.______ on Jun. 2, 1995.

[0022] A polynucleotide encoding a polypeptide of the present inventionwas discovered in a cDNA library derived from human 9 week embryo. It isstructurally related to the CCN family. It contains an open readingframe encoding a protein of 206 amino acid residues of which the first23 amino acids residues are the putative leader sequence such that themature protein comprises 183 amino acids.

[0023] The designation of SCGF as a member of the CCN growth factorfamily was based primarily through conservation of amino acid sequences.The homology to the CCN family is approximately 27% with all themembers. The highest similarity to CCN members is 62% over a 29 aminoacid stretch of SCGF. The polypeptide of the present invention iscysteine-rich with 10 cysteines. It is about 50% the size of CTGF, cyr61and nov.

[0024] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIG. 1 (SEQ ID NO: 1) or thatof the deposited clone or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptide as the DNA of FIG. 1(SEQ ID NO:1) or the deposited cDNA.

[0025] The polynucleotide which encodes for the mature polypeptide ofFIG. 1 (SEQ ID No. 2) or for the mature polypeptide encoded by thedeposited cDNA may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

[0026] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0027] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIG. 1 (SEQ ID No. 2) or the polypeptide encoded by the cDNAof the deposited clone. The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

[0028] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIG. 1 (SEQ ID No. 2) or the samemature polypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID No. 2) or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

[0029] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

[0030] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.once the prosequence is cleaved an active mature protein remains.

[0031] Thus, for example, the polynucleotide of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and a presequence (leadersequence).

[0032] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian hosto e.g., COS-7cells, is used. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767(1984)).

[0033] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s), i.e. function as an SCGF polypeptide.

[0034] Alternatively, the polynucleotides may have at least 20 bases,preferably 30 bases and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which have anidentity thereto, as hereinabove described, and which does not retainactivity. Such polynucleotides may be employed as probes for thepolynucleotide of SEQ ID NO:1, or for variantb thereof, for example, forrecovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

[0035] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of still in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0036] The present invention further relates to a polypeptide which hasthe deduced amino acid sequence of FIG. 1 (SEQ ID No:2) or which has theamino acid sequence encoded by the deposited cDNA, as well as fragments,analogs and derivatives of such polypeptide.

[0037] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1 (SEQ ID No. 2) or that encoded by thedeposited cDNA, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0038] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0039] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID No. 2) or that encoded by the deposited cDNA may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence or (v) splice variants of themature polypeptide which are lacking certain amino acid residues yetstill retain biological activity. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0040] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0041] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0042] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0043] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0044] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0045] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0046] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the SCGF genes. The culture conditions, suchas temperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

[0047] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0048] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0049] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli, lac or trp, the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome-binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0050] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0051] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0052] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

[0053] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pDlO, phagescript, psiXl74, pbluescript SK, PBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK2233, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: PWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, PBPV, PMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0054] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PRI PL andtrp. Bukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0055] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a ma lian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAEDextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0056] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0057] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0058] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0059] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0060] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomanas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0061] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMI (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0062] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0063] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents, such methods are well know to those skilled in the art.

[0064] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0065] The polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the mature protein.Finally, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

[0066] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0067] The polypeptide of the present invention may be employed inwound-healing and associated therapies concerned with re-growth oftissue, such as connective tissue, skin, bone, cartilage, muscle, lungor kidney.

[0068] The polypeptide of the present invention may be employed fortissue remodeling such as restenosis, cardiac dilation/hypertrophy(congestive heart failure) and atherosclerosis.

[0069] The polypeptide may also be employed to stimulate angiogenesis,for example, to enhance the growth of vascular smooth muscle andendothelial cells. The increase in angiogenesis would be beneficial toischemic tissues and to collateral coronary development in the heartsubsecluent to coronary stenosis.

[0070] The polypeptide of the present invention may also be employedduring implant fixation to stimulate the growth of cells around theimplant and therefore, facilitate its attachment to its intended site.

[0071] The polypeptide of the present invention may be employed tostimulate early growth of an embryo, since the expression pattern of theSCGF polypeptide is abundant in embryonic libraries.

[0072] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, as a research reagent for invitro purposes related to scientific research, synthesis of DNA andmanufacture of DNA vectors, for the purpose of developing therapeuticsand diagnostics for the treatment of human disease.

[0073] This invention provides a method for identification of thereceptor for the polypeptide of the present invention. The gene encodingthe receptor can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to SCGF polypeptides, and a cDNA librarycreated from this RNA is divided into pools and used to transfect COScells or other cells that are not responsive to SCGF. Transfected cellswhich are grown on glass slides are exposed to labeled SCGF. SCGF can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase. Following fixationand incubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared and retransfected using an iterative sub-pooling and rescreening process, eventuallyyielding a single clone that encodes the putative receptor.

[0074] As an alternative approach for receptor identification, labeledSCGF can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the SCGF-receptor can be excised, resolved into peptidefragments, and subjected to protein microsequencing. The amino acidsequence obtained from microsequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

[0075] This invention is also related to a method of screening compoundsto identify those which bind to and activate the SCGF receptors. Anexample of such a method takes advantage of the ability of SCGF tostimulate the proliferation of endothelial cells in the presence of thecomitogen Con A. Human umbilical vein endothelial cells are obtained andcultured in 96-well flat-bottomed culture plates (Costar, Cambridge,Mass.) and supplemented with a reaction mixture appropriate forfacilitating proliferation of the cells, the mixture containing Con-A(Calbiochem, La Jolla, Calif.). Con-A and the compound to be screenedare added and after incubation at 37° C., cultures are pulsed with³[H]thymidine and harvested onto glass fiber filters (PhD; CambridgeTechnology, Watertown, Mass.). Mean ³[H]-thymidine incorporation (cpm)of triplicate cultures is determined using a liquid scintillationcounter (Beckman Instruments, Irvine, Calif.). Significants³[H]-thymidine incorporation indicates stimulation of endothelial cellproliferation.

[0076] To assay for antagonists, the assay described above is performed,however, in this assay SCGF is added along with the compound to bescreened and the ability of the compound to inhibit ³ [H]-thymidineincorporation in the presence of SCGF, indicates that the compound is anantagonist to SCGF. Alternatively, SCGF antagonists may be detected bycombining SCGF and a potential antagonist with membrane-bound SCGFreceptors or recombinant receptors under appropriate conditions for acompetitive inhibition assay. SCGF can be labeled, such as byradioactivity, such that the number of SCGF molecules bound to thereceptor can determine the effectiveness of the potential antagonist.

[0077] Examples of potential SCGF antagonists include an antibody, or insome cases, an oligonucleotide, which binds to the polypeptide.Alternatively, a potential antagonist may be a closely related protein,for example, a mutated form of SCGF, which recognizes the SCGF receptorbut imparts no effect, thereby competitively inhibiting the action ofSCGF.

[0078] Another potential SCGF antagonist is an antisense constructprepared using antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptides of the presentinvention, is used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcription(triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney etal, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360(1991)), thereby preventing transcription and the production of SCGF.The antisense RNA oligonucleotide hybridizes to the mRNA in vivo andblocks translation of the mRNA molecule into the SCGF (antisense—Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of SCGF.

[0079] Potential SCGF antagonists include small molecules which bind tothe active site, the receptor binding site, or other growth factorbinding site of the polypeptide thereby blocking the normal biologicalactivity of SCGF. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

[0080] The antagonists may be employed to inhibit tumorneovascularization and the neointimal proliferation of smooth musclecells prevalent in atherosclerosis and restenosis subsequent to balloonangioplasty.

[0081] The antagonists may also be employed to inhibit the overproduction of scar tissue seen in a keloid which forms after surgery,fibrosis after myocardial infarction, or fibrotic lesions associatedwith pulmonary fibrosis. The antagonists may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., ashereinafter described.

[0082] The SCGF polypeptides and antagonist or agonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier. Such compositions comprise a therapeutically effective amountof the polypeptide, agonist and antagonist and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration.

[0083] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions may be employed in conjunctionwith other therapeutic compounds.

[0084] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, The pharmaceuticalintranasal or intradermal routes. The pharmaceutical compositions areadministered in an amount which is effective for treating and/orprophylaxis of the specific indication. In general, they areadministered in an amount of at least about 10 μg/kg body weight and inmost cases they will be administered in an amount not in excess of about8 mg/Kg body weight per day. In most cases, the dosage is from about 10μg/kg to about 1 mg/kg body weight daily, taking into account the routesof administration, symptoms, etc.

[0085] SCGF in combination with other growth factors including but notlimited to, PDGF, IGF, FGF, EGF or TGF-β may accelerate physiologicalresponses as seen in wound healing.

[0086] The SCGF polypeptide and agonists and antagonists which arepolypeptides, may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

[0087] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0088] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0089] Retroviruses from which the retroviral plasmed vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0090] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0091] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe genes encoding the polypeptides.

[0092] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAml2, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO4precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0093] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblaStE,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0094] This invention is also related to the use of the gene of thepresent invention as a diagnostic. Detection of a mutation in thenucleic acid sequence encoding a polypeptide of the present inventionwill allow a diagnosis of a disease or a susceptibility to a disease,such as a tumor, since mutations in SCGF may cause tumors.

[0095] Individuals carrying mutations in the human SCGF gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding SCGF can be used toidentify and analyze SCGF mutations. For example, deletions andinsertions can be detected by a change in size of the amplif ied productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled SCGF RNA or alternatively,radiolabeled SCGF antisense DNA sequences. Perfectly matched sequencescan be distinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0096] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0097] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0098] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0099] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0100] SCGF protein expression may be linked to vascular disease orneovascularization associated with tumor formation. SCGF may haveoncogenic effects on kidney, lung, heart or brain. Tissues in embryosare rapidly growing and an SCGF specific antibody could detect tumorswhich grow quickly. SCGF has a signal peptide and the mRNA is highlyexpressed in endothelial cells and to a lesser extent in smooth musclecells. Accordingly, an anti-SCGF antibody could be used to diagnosevascular disease or neovascularization associated with tumor formationsince an altered level of this polypeptide may be indicative of suchdisorders. Further, the SCGF protein is thought to be involved in tissueremodeling processes such as restenosis, hypertension, congestive heartfailure and atherosclerosis.

[0101] A competition assay may be employed wherein antibodies specificto SCGF are attached to a solid support and labeled SCGF and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of SCGF in the sample.

[0102] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay the SCGF polypeptide is passed over a solid support and binds toantibody attached to a solid support. A second antibody is then bound tothe SCGF polypeptide. A third antibody which is labeled and specific tothe second antibody is then passed over the solid support and binds tothe second antibody and an amount can then be quantified.

[0103] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0104] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 31untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0105] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0106] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases. For a review of this technique, see Vermaet al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0107] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0108] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0109] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0110] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0111] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptider, into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0112] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0113] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimunogenic polypeptide products of this invention.

[0114] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0115] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0116] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0117] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmed or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0118] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0119] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0120] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0121] Unless otherwise stated, transf ormation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1

[0122] Bacterial Expression and Purification of SCGF

[0123] The DNA sequence encoding SCGF, ATCC was initially amplifiedusing PCR oligonucleotide primers corresponding to the 5′ sequences ofthe processed SCGF protein (minus the signal peptide sequence) and thevector sequences 3′ to the SCGF gene. Additional nucleotidescorresponding to SCGF were added to the 5′ and 3′ sequencesrespectively. The 5′ oligonucleotide primer has the sequence 5′CACTGCAAGCTTATTTAAAAATGATGCCACAGAA 3′ (SEQ ID NO:3) contains a Hind IIIrestriction enzyme site (in bold) followed by 21 nucleotides of SCGFcoding sequence starting from the presumed terminal amino acid of theprocessed protein codon (underlined). The 3′ oligonucleotide primer 5′CATGCCTCTAGATATGGGAGTCTGAGTTCTAAC 3′ (SEQ ID NO:4) contains an Xba Irestriction site (in bold) followed by the reverse complement ofnucleotides corresponding to the carboxy terminal 5 amino acids and thetranslational stop codon (underlined). The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE9 (Qiagen, Inc. Chatsworth, Calif.,). pQE-9 encodes antibioticresistance (Ampr), a bacterial origin of replication (ori), anIPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS),a 6-His tag and restriction enzyme sites. pQE-9 and the SCGF PCR productwere then digested with Hind III and Xba I and ligated together with T4DNA ligase. The desired recombinants would contain the SCGF codingsequence inserted downstream from the histidine tag and the ribosomebinding site. The ligation mixture was then used to transform E. colistrain M15 [pREP5] (Qiagen, Inc.) by the procedure described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989). M15[pREP5] contains multiple copies of theplasmid pREP5, which expresses the laci repressor and also conf erskanamycin resistance (Kan^(r)). Transforments were identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and conf irmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The OIN culture was used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the laci repressor,clearing the P/0 leading to increased gene expression. Cells were grownan extra 3 to 4 hours such that there is an exponential growth culturepresent. Cells were then harvested by centrifugation. TheSCGF/6-Histidine-containing M15 [pREP4] cells were lysed in 6M GnHC1, 50mM NaPO4 at pH 8.0. The lysate was loaded on a Nickel-Chelate column andthe flow-through collected. The column was washed with 6M GnHC1, 50 mMNapo4at pH 8.0, 6.0 and 5.0. The SCGF fusion protein (>90% pure) waseluted at pH 2.0. For the purpose of renaturation, the pH 2.0 eluate wasadjusted to 3 molar guanidine HC1, 100 mm sodium phosphate, 10 mmolarglutathione (reduced) and 2 mmolar glutathione (oxidized). Afterincubation in this solution for 12 hours the protein was dialyzed to 10remolar sodium phosphate. To run the gel, the pellets were resuspendedin SDS/NAOH and SDS-PAGE loading buffer, heat denatured, thenelectrophoresed on a 15% denaturing polyacrylamide gel. The proteinswere visualized with Coomassie Brilliant Blue R-250 stain.

EXAMPLE 2

[0124] Cloning and Expression of SCGF Using the Baculovirus ExpressionSystem

[0125] The DNA sequence encoding SCGF, ATCC was initially amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the SCGF coding region. Additional nucleotidescorresponding to SCGF were added to the 5′ and 3′ sequencesrespectively. The 5′ oligonucleotide primer has the sequence 5′CATTCGCGGATCCBCCATCATGCTTCCTCCTGCCATTCAT 3′ (SEQ ID NO:3) contains aBamHI restriction enzyme site (in bold) followed by 21 nucleotides ofSCGF coding sequence starting from the presumed initiating methionine ofthe unprocessed protein (underlined). The 3′ oligonucleotide primer 5′CACTGCCTCTAGATATGGGAGTCTGAGTTCTAAC 3′ (SEQ ID NO:4) contains an Xba Irestriction site followed by the reverse complement of nucleotidescorresponding to the 16 3′ untranslated nucleotides adjacent to thetranslational stop codon (underlined). The restriction enzyme sitescorrespond to the restriction enzyme sites on the baculovirus expressionvector pA2 (Qiagen, Inc. Chatsworth, Calif.). The SCGF PCR product andpA2 were then digested with BamHI and Xba I and ligated together with T4DNA ligase.

[0126] The sequence of the cloned fragment is confirmed by DNAsequencing.

[0127] 5 ug of the plasmid pbac SCGF is cotransfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Feloner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0128] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac SCGFare mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg,Md.)—Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added,mixed and incubated for 15 minutes at room temperature. Then thetransfection mixture is added dropwise to the Sf9 insect cells (ATCC CRL1711) seeded in a 35 mm tissue culture plate with 1 ml Grace' mediumwithout serum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 20° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate is put back into an incubator and cultivation continued at 27°C. for four days.

[0129] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal”(Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0130] Four days after the serial dilution, the viruses are added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculoviruses is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0131] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-SCGF at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 3

[0132] Expression of Recombinant SCGF in CHO Cells

[0133] The vector pN346 is used for the expression of the SCGF protein.Plasmid pN346 is a derivative of the plasmid pSV2-dhfr [ATCC AccessionNo. 37146). Both plasmids contain the mouse dhfr gene under control ofthe SV40 early promoter. Chinese hamster ovary or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Lift Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplication of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. i. and Sydenham, M. A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe dhfr gene it is usually co-amplified and overexpressed.Subsequently, when the methotrexate is withdrawn, cell lines contain theamplified gene integrated into the chromosome(s).

[0134] Plasmid pN346 contains for the expression of the gene of interesta strong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March 1985,438-447) plus a fragment isolated from the enhancer of the immediateearly gene of human cytomegalovirus (CMV) (Boshart et al., Cell41:521-530, 1985). Downstream of the promoter are the following singlerestriction enzyme cleavage sites that allow the integration of thegenes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmidcontains translational stop codons in all three reading frames followedby the 3′ intron and the polyadenylation site of the rat preproinsulingene. Other high efficient promoters can also be used for theexpression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.

[0135] Stable cell lines carrying a gene of interest integrated into thechromosome can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g. G418 plusmethotrexate.

[0136] The plasmid pN346 is digested with the restriction enzyme BamHIand then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

[0137] The DNA sequence encoding the full length SCGF protein, ATCC #_______, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

[0138] The 5′ primer has the sequence 5′CATCGCGGATCCGCCATCATGCTTCCTCCTGCCATTCAT 3′ and contains a BamHIrestriction enzyme site (in bold) followed by 21 nucleotides resemblingan efficient signal for the initiation of translation in eukaryoticcells (Kozak, M., J. Mol. Biol., 196:947-950, (1987)). The remainingnucleotides correspond to the amino terminal 7 amino acids including thetranslational initiation codon (underlined). The 3′ primer has thesequence 5′ CAC TGCGGATCCTATGGGAGTCTGAGTTCTAAC 3′ and contains a BamHIrestriction site (in bold) and 21 nucleotides that are the reversecomplement of 3′ untranslated DNA starting 16 nucleotides downstreamfrom the translational stop codon. The PCR product is digested withBamHI and purified on a 1% agarose gel using a commercially availablekit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). This fragment is thenligated to BamHI digested, phosphatased pN346 plasmid with T4 DNAligase. XllBlue (Stratagene) E. coli are transformed and plated on LB,50 μg/ml ampicillin plates. Colonies bearing the desired recombinant inthe proper orientation are screened for by PCR with a 5′ primer whichcorresponds to the Rous sarcoma virus promoter and a 3′ primer whichcorresponds to the reverse complement of SCGF codons 73-79. The sequenceof the cloned fragment is confirmed by DNA sequencing.

[0139] Transfection of CHO-dhfr-cells

[0140] Chinese hamster ovary cells lacking an active DHFR enzyme areused for transfection. 5 μg of the expression plasmid pN346SCGF arecotransfected with 0.5 μg of the plasmid pSVneo using the lipofectinmethod (Feloner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the gene neo from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) and cultivated from 10-14 days. After this period,single clones are trypsinized and then seeded in 6-well petri dishesusing different concentrations of methotrexate (25, 50 nm, 100 nm, 200nm, 400 nm). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). Thesame procedure is repeated until clones grew at a concentration of 100μM.

[0141] The expression of the desired gene product is analyzed by Westernblot analysis and SDS-PAGE.

EXAMPLE 4

[0142] Tissue Localization of SCGF Gene Expression by Northern BlotAnalysis

[0143] A multiple tissue Northern blot (Clontech Laboratories, Inc.,4030 Fabian Way; Palo Alto, Calif. 94303) containing 2 ug of human adultbrain, heart, placenta, lung, liver skeletal muscle, kidney, andpancreas poly A+ mRNA per lane is prehybridized in Church buffer(Church, G. M. Gilbert, W. , Proc. Natl. Acad. Sci. USA 81, 1991-1995(1984) at 60° C. for one hour. The DNA sequence coding for SCGF, ATCC#______, is amplified from the full length cDNA cloned in pBluescriptSK(−) using the M13 Forward (5′ GTAA AACGACGGCCAGT 3′) and Reverse (5′GGAAACAGCTATGACCATG 3′) primers. Twenty-five nanograms of PCR product israndom primer radiolabeled (Prime-It II, Stratagene Cloning Systems,11011 North Torrey Pines Rd.; La Jolla, Calif. 92037) with ³²P-dCTP. Theheat denatured SCGF probe is added directly to the prehybridizationbuffer and incubated 16 hr at 60° C. Two ten minute washes are performedin 0.2×SSC, 0.1% SDS at 60° C. Autoradiography is performed at −80° C.

EXAMPLE 5

[0144] Expression via Gene Therapy

[0145] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0146] pKV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988)) flanked bythe long terminal repeats of the moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0147] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer having contains a HindIII site. Equal quantities of theMoloney reurine sarcoma virus linear backbone and the EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HE101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interebtproperly inserted.

[0148] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbeccols Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0149] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quiqklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0150] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0151] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 20 900 BASE PAIRS NUCLEIC ACID SINGLE LINEAR cDNA 1 CGCCAAACCTCTATGGATAT ATAAAGGGAA GCTTGAGGAG GAATTTCACA GTTACAGTGC 60 AGAAGCAGAGGCAAAAGAAT TAACCAGCTC TTCAGTCAAG CAAATCCTCT ACTCACCATG 120 CTTCCTCCTGCCATTCATTT CTATCTCCTT CCCCTTGCAT GCATCCTAAT GAAAAGCTGT 180 TTGGCTTTTAAAAATGATGC CACAGAAATC CTTTATTCAC ATGTGGTTAA ACCTGTTCCA 240 GCACACCCCAGCAGCAACAG CACGTTGAAT CAAGCCAGAA ATGGAGGCAG GCATTTCAGT 300 AACACTGGACTGGATCGGAA CACTCGGGTT CAAGTGGGTT GCCGGGAACT GCGTTCCACC 360 AAATACATCTCTGATGGCCA GTGCACCAGC ATCAGCCCTC TGAAGGAGCT GGTGTGTGCT 420 GGCGAGTGCTTGCCCCTGCC AGTGCTCCCT AACTGGATTG GAGGAGGCTA TGGAACAAAG 480 TACTGGAGCAGGAGGAGCTC CCAGGAGTGG CGGTGTGTCA ATGACAAAAC CCGTACCCAG 540 AGAATCCAGCTGCAGTGCCA AGATGGCAGC ACACGCACCT ACAAAATCAC AGTAGTCACT 600 GCCTGCAAGTGCAAGAGGTA CACCCGGCAG CACAACGAGT CCAGTCACAA CTTTGAGAGC 660 ATGTCACCTGCCAAGCCAGT CCAGCATCAC AGAGAGCGGA AAAGAGCCAG CAAATCCAGC 720 AAGCACAGCATGAGTTAGAA CTCAGACTCC CATAACTAGA CTTACTAGTA ACCATCTGCT 780 TTACAGATTTGATTGCTTGG AAGACTCAAG CCTGCCACTG CTGTTTTCTC ACTTGAAAGT 840 ATATGCTTTCTGCTTTGATC AAACCCAGCA AGCTGTCTTA AGTATCAGGA CCTTCTTTGG 900 206 AMINOACIDS AMINO ACID <Unknown> LINEAR PROTEIN 2 Met Lys Pro Pro Ala Ile HisPhe Tyr Leu Leu Pro Leu Ala Cys -20 -15 -10 Ile Leu Met Lys Ser Cys LeuAla Phe Lys Asn Asp Ala Thr Glu -5 1 5 Ile Leu Tyr Ser His Val Val LysPro Val Pro Ala His Pro Ser 10 15 20 Ser Asn Ser Thr Leu Asn Gln Ala ArgAsn Gly Gly Arg His Phe 25 30 35 Ser Asn Thr Gly Leu Asp Arg Asn Thr ArgVal Gln Val Gly Cys 40 45 50 Arg Glu Leu Arg Ser Thr Lys Tyr Ile Ser AspGly Gln Cys Thr 55 60 65 Ser Ile Ser Pro Leu Lys Glu Leu Val Cys Ala GlyGlu Cys Leu 70 75 80 Pro Leu Pro Val Leu Pro Asn Trp Ile Gly Gly Gly TyrGly Thr 85 90 95 Lys Tyr Trp Ser Arg Arg Ser Ser Gln Glu Trp Arg Cys ValAsn 100 105 110 Asp Lys Thr Arg Thr Gln Arg Ile Gln Leu Gln Cys Gln AspGly 115 120 125 Ser Thr Arg Thr Tyr Lys Ile Thr Val Val Thr Ala Cys LysCys 130 135 140 Lys Arg Tyr Thr Arg Gln His Asn Glu Ser Ser His Asn PheGlu 145 150 155 Ser Met Ser Pro Ala Lys Pro Val Gln His His Arg Glu ArgLys 160 165 170 Arg Ala Ser Lys Ser Ser Lys His Ser Met Ser 175 180 34base pairs nucleic acid not relevant linear DNA (genomic) 3 CACTGCAAGCTTATTTAAAA ATGATGCCAC AGAA 34 33 base pairs nucleic acid not relevantlinear DNA (genomic) 4 CATGCCTCTA GATATGGGAG TCTGAGTTCT AAC 33 39 basepairs nucleic acid not relevant linear DNA (genomic) 5 CATTCGCGGATCCCCATCAT GCTTCCTCCT GCCATTCAT 39 34 base pairs nucleic acid notrelevant linear DNA (genomic) 6 CACTGCCTCT AGATATGGGA GTCTGAGTTC TAAC 3439 base pairs nucleic acid not relevant linear DNA (genomic) 7CATCGCGGAT CCGCCATCAT GCTTCCTCCT GCCATTCAT 39 30 base pairs nucleic acidnot relevant linear DNA (genomic) 8 TGCGGATCCT ATGGGAGTCT GAGTTCTAAC 3017 base pairs nucleic acid not relevant linear DNA (genomic) 9GTAAAACGAC GGCCAGT 17 19 base pairs nucleic acid not relevant linear DNA(genomic) 10 GGAAACAGCT ATGACCATG 19 379 AMINO ACIDS AMINO ACID<Unknown> LINEAR PROTEIN 11 Met Ser Ser Ser Thr Phe Arg Thr Leu Ala ValAla Val Thr Leu 5 10 15 Leu His Leu Thr Arg Leu Ala Leu Ser Thr Cys ProAla Ala Cys 20 25 30 His Cys Pro Leu Glu Ala Pro Lys Cys Ala Pro Gly ValGly Leu 35 40 45 Val Arg Asp Gly Cys Gly Cys Cys Lys Val Cys Ala Lys GlnLeu 50 55 60 Asn Glu Asp Cys Ser Lys Thr Gln Pro Cys Asp His Thr Lys Gly65 70 75 Leu Glu Cys Asn Phe Gly Ala Ser Ser Thr Ala Leu Lys Gly Ile 8085 90 Cys Arg Ala Gln Ser Glu Gly Arg Pro Cys Glu Tyr Asn Ser Arg 95 100105 Ile Tyr Gln Asn Gly Glu Ser Phe Gln Pro Asn Cys Lys His Gln 110 115120 Cys Thr Cys Ile Asp Gly Ala Val Gly Cys Ile Pro Leu Cys Pro 125 130135 Gln Glu Leu Ser Leu Pro Asn Leu Gly Cys Pro Asn Pro Arg Leu 140 145150 Val Lys Val Ser Gly Gln Cys Cys Glu Glu Trp Val Cys Asp Glu 155 160165 Asp Ser Ile Lys Asp Ser Leu Asp Asp Gln Asp Asp Leu Leu Gly 170 175180 Leu Asp Ala Ser Glu Val Glu Leu Thr Arg Asn Asn Glu Leu Ile 185 190195 Ala Ile Gly Lys Gly Ser Ser Leu Lys Arg Leu Pro Val Phe Gly 200 205210 Thr Glu Pro Arg Val Leu Phe Asn Pro Leu His Ala His Gly Gln 215 220225 Lys Cys Ile Val Gln Thr Thr Ser Trp Ser Gln Cys Ser Lys Ser 230 235240 Cys Gly Thr Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Pro Glu 245 250255 Cys Arg Leu Val Lys Glu Thr Arg Ile Cys Glu Val Arg Pro Cys 260 265270 Gly Gln Pro Val Tyr Ser Ser Leu Lys Lys Gly Lys Lys Cys Ser 275 280285 Lys Thr Lys Lys Ser Pro Glu Pro Val Arg Phe Thr Tyr Ala Gly 290 295300 Cys Ser Ser Val Lys Lys Tyr Arg Pro Lys Tyr Cys Gly Ser Cys 305 310315 Val Asp Gly Arg Cys Cys Thr Pro Leu Gln Thr Arg Thr Val Lys 320 325330 Met Arg Phe Arg Cys Glu Asp Gly Glu Met Phe Ser Lys Asn Val 335 340345 Met Met Ile Gln Ser Cys Lys Cys Asn Tyr Asn Cys Pro His Pro 350 355360 Asn Glu Ala Ser Phe Arg Leu Tyr Ser Leu Phe Asn Asp Ile His 365 370375 Lys Phe Arg Asp 374 AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN12 Met Ser Ser Arg Ile Val Arg Glu Leu Ala Leu Val Val Thr Leu 5 10 15Leu His Leu Thr Arg Val Gly Leu Ser Thr Cys Pro Ala Asp Cys 20 25 30 HisCys Pro Leu Glu Ala Pro Lys Cys Ala Pro Gly Val Gly Leu 35 40 45 Val ArgAsp Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gln Leu 50 55 60 Asn Glu AspCys Arg Lys Thr Gln Pro Cys Asp His Thr Lys Gly 65 70 75 Leu Glu Cys AsnPhe Gly Ala Ser Ser Thr Ala Leu Lys Gly Ile 80 85 90 Cys Arg Ala Gln SerGlu Gly Arg Pro Cys Glu Tyr Asn Ser Arg 95 100 105 Ile Tyr Gln Asn GlyGlu Ser Phe Gln Pro Asn Cys Lys His Gln 110 115 120 Cys Thr Cys Ile GlyTrp Arg Arg Gly Ala Cys Ile Pro Leu Cys 125 130 135 Pro Gln Glu Leu SerLeu Pro Asn Leu Gly Cys Pro Asn Pro Arg 140 145 150 Leu Val Lys Val ThrGly Gln Cys Cys Glu Glu Trp Val Cys Asp 155 160 165 Glu Asp Ser Ile LysAsp Pro Met Glu Asp Gln Asp Gly Leu Leu 170 175 180 Gly Lys Gly Leu GlyPhe Asp Ala Ser Glu Val Glu Leu Thr Arg 185 190 195 Asn Asn Glu Leu IleAla Val Gly Lys Gly Ser Ser Leu Lys Arg 200 205 210 Leu Pro Val Phe GlyMet Glu Pro Arg Ile Leu Tyr Asn Pro Leu 215 220 225 Gln Gly Gln Lys CysIle Val Gln Thr Thr Ser Trp Ser Gln Cys 230 235 240 Ser Lys Thr Cys GlyThr Gly Ile Ser Thr Arg Val Thr Asn Asp 245 250 255 Asn Pro Glu Cys ArgLeu Val Lys Glu Thr Arg Ile Cys Glu Val 260 265 270 Arg Pro Cys Gly GlnPro Val Tyr Ser Ser Leu Lys Lys Gly Lys 275 280 285 Lys Cys Ser Lys ThrLys Lys Ser Pro Glu Pro Val Arg Phe Thr 290 295 300 Tyr Ala Gly Cys LeuSer Val Lys Lys Tyr Arg Pro Lys Tyr Cys 305 310 315 Gly Ser Cys Val AspGly Arg Cys Cys Thr Pro Gln Leu Thr Arg 320 325 330 Thr Val Lys Met ArgPhe Pro Cys Glu Asp Gly Glu Thr Phe Ser 335 340 345 Lys Asn Val Met MetIle Gln Ser Ser Lys Cys Asn Tyr Asn Cys 350 355 360 Pro His Ala Asn GluAla Ala Phe Pro Phe Tyr Arg Leu Phe 365 370 375 AMINO ACIDS AMINO ACID<Unknown> LINEAR PROTEIN 13 Met Gly Ser Ala Gly Ala Arg Pro Ala Leu AlaAla Ala Leu Leu 5 10 15 Cys Leu Ala Arg Leu Ala Leu Gly Ser Pro Cys ProAla Val Cys 20 25 30 Gln Cys Pro Ala Ala Ala Pro Gln Cys Ala Pro Gly ValGly Leu 35 40 45 Val Pro Asp Gly Cys Gly Cys Cys Lys Val Cys Ala Lys GlnLeu 50 55 60 Asn Glu Asp Cys Ser Arg Thr Gln Pro Cys Asp His Thr Lys Gly65 70 75 Leu Glu Cys Asn Phe Gly Ala Ser Pro Ala Ala Thr Asn Gly Ile 8085 90 Cys Arg Ala Gln Ser Glu Gly Arg Pro Cys Glu Tyr Asn Ser Lys 95 100105 Ile Tyr Gln Asn Gly Glu Ser Phe Gln Pro Asn Cys Lys His Gln 110 115120 Cys Thr Cys Ile Asp Gly Ala Val Gly Cys Ile Pro Leu Cys Pro 125 130135 Gln Glu Leu Ser Leu Pro Asn Leu Gly Cys Pro Ser Pro Arg Leu 140 145150 Val Lys Val Pro Gly Gln Cys Cys Glu Glu Trp Val Cys Asp Glu 155 160165 Ser Lys Asp Ala Leu Glu Glu Leu Glu Gly Phe Phe Ser Lys Glu 170 175180 Phe Gly Leu Asp Ala Ser Glu Gly Glu Leu Thr Arg Asn Asn Glu 185 190195 Leu Ile Ala Ile Val Lys Gly Gly Leu Lys Met Leu Pro Val Phe 200 205210 Gly Ser Glu Pro Gln Ser Arg Ala Phe Glu Asn Pro Lys Cys Ile 215 220225 Val Gln Thr Thr Ser Trp Ser Gln Cys Ser Lys Thr Cys Gly Thr 230 235240 Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Pro Asp Cys Lys Leu 245 250255 Ile Lys Glu Thr Arg Ile Cys Glu Val Arg Pro Cys Gly Gln Pro 260 265270 Ser Tyr Ala Ser Leu Lys Lys Gly Lys Lys Cys Thr Lys Thr Lys 275 280285 Lys Ser Pro Ser Pro Val Arg Phe Thr Tyr Ala Gly Cys Ser Ser 290 295300 Val Lys Lys Tyr Arg Pro Lys Tyr Cys Gly Ser Cys Val Asp Gly 305 310315 Arg Cys Cys Thr Pro Gln Gln Thr Arg Thr Val Lys Ile Arg Phe 320 325330 Arg Cys Asp Asp Gly Glu Thr Phe Thr Lys Ser Val Met Met Ile 335 340345 Gln Ser Cys Arg Cys Asn Tyr Asn Cys Pro His Ala Asn Glu Ala 350 355360 Tyr Pro Phe Tyr Arg Leu Val Asn Asp Ile His Lys Phe Arg Asp 365 370375 348 AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN 14 Met Thr AlaAla Ser Met Gly Pro Val Arg Val Ala Phe Val Val 5 10 15 Leu Leu Ala LeuCys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser 20 25 30 Gly Pro Cys Arg CysPro Asp Glu Pro Ala Pro Arg Cys Pro Ala 35 40 45 Gly Val Ser Leu Val AspGly Cys Gly Cys Cys Arg Val Cys Ala 50 55 60 Lys Gln Leu Gly Glu Leu CysThr Glu Arg Asp Pro Cys Asp Pro 65 70 75 His Lys Gly Leu Phe Cys Asp PheGly Ser Pro Ala Asn Arg Lys 80 85 90 Ile Gly Val Cys Thr Ala Lys Asp GlyAla Pro Cys Ile Phe Gly 95 100 105 Gly Thr Val Tyr Arg Ser Gly Glu SerPhe Gln Ser Ser Cys Lys 110 115 120 Tyr Gln Cys Thr Cys Leu Asp Gly AlaVal Gly Cys Met Pro Leu 125 130 135 Cys Ser Met Asp Val Arg Leu Pro SerPro Asp Cys Pro Phe Pro 140 145 150 Arg Arg Val Lys Leu Pro Gly Lys CysCys Glu Glu Trp Val Cys 155 160 165 Asp Glu Pro Lys Asp Gln Thr Val ValGly Pro Ala Leu Ala Ala 170 175 180 Tyr Arg Leu Glu Asp Thr Phe Gly ProAsp Pro Thr Met Ile Arg 185 190 195 Ala Asn Cys Leu Val Gln Thr Thr GluTrp Ser Ala Cys Ser Lys 200 205 210 Thr Cys Gly Met Gly Ile Ser Thr ArgVal Thr Asn Asp Asn Ala 215 220 225 Ser Cys Arg Leu Glu Lys Gln Ser ArgLeu Cys Met Val Arg Pro 230 235 240 Cys Glu Ala Asp Leu Glu Glu Asn IleLys Lys Gly Lys Lys Cys 245 250 255 Ile Arg Thr Pro Lys Ile Ser Lys ProIle Lys Phe Glu Leu Ser 260 265 270 Gly Cys Thr Ser Met Lys Thr Tyr ArgAla Lys Phe Cys Gly Val 275 280 285 Cys Thr Asp Gly Arg Cys Cys Thr ProHis Arg Thr Thr Thr Leu 290 295 300 Pro Val Glu Phe Lys Cys Pro Asp GlyGlu Val Met Lys Lys Asn 305 310 315 Met Met Phe Ile Lys Thr Cys Ala CysHis Tyr Asn Cys Pro Gly 320 325 330 Asp Asn Asp Ile Phe Glu Ser Leu TyrTyr Arg Lys Met Tyr Gly 335 340 345 Asp Met Ala 348 AMINO ACIDS AMINOACID <Unknown> LINEAR PROTEIN 15 Met Leu Ala Ser Val Ala Gly Pro Ile SerLeu Ala Leu Val Leu 5 10 15 Leu Ala Leu Cys Thr Arg Pro Ala Thr Gly GlnAsp Cys Ser Ala 20 25 30 Gln Cys Gln Cys Ala Ala Glu Ala Ala Pro His CysPro Ala Gly 35 40 45 Val Ser Leu Val Leu Asp Gly Cys Gly Cys Cys Arg ValCys Ala 50 55 60 Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys AspPro 65 70 75 His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg Lys80 85 90 Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Val Phe Gly 95100 105 Gly Ser Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys 110115 120 Tyr Gln Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Val Pro Leu 125130 135 Cys Ser Met Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro 140145 150 Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Lys Glu Trp Val Cys 155160 165 Asp Glu Pro Lys Asp Arg Thr Ala Val Gly Pro Ala Leu Ala Ala 170175 180 Tyr Arg Leu Glu Asp Thr Phe Gly Pro Asp Pro Thr Met Met Arg 185190 195 Ala Asn Cys Leu Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys 200205 210 Thr Cys Gly Met Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Thr 215220 225 Phe Cys Arg Leu Glu Lys Gln Ser Arg Leu Cys Met Val Arg Pro 230235 240 Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys Gly Lys Lys Cys 245250 255 Ile Arg Thr Pro Lys Ile Ala Lys Pro Val Lys Phe Glu Leu Ser 260265 270 Gly Cys Thr Ser Val Lys Thr Tyr Arg Ala Lys Phe Cys Gly Val 275280 285 Cys Thr Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr Thr Leu 290295 300 Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Ile Met Lys Lys Asn 305310 315 Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gly 320325 330 Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly 335340 345 Asp Met Ala 351 AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN16 Met Glu Thr Gly Gly Gly Gln Gly Leu Pro Val Leu Leu Leu Leu 5 10 15Leu Leu Leu Leu Arg Pro Cys Glu Val Ser Gly Arg Glu Ala Ala 20 25 30 CysPro Arg Pro Cys Gly Gly Arg Cys Pro Ala Glu Pro Pro Arg 35 40 45 Cys AlaPro Gly Val Pro Ala Val Leu Asp Gly Cys Gly Cys Cys 50 55 60 Leu Val CysAla Arg Gln Arg Gly Glu Ser Cys Ser Pro Leu Leu 65 70 75 Pro Cys Asp GluSer Gly Gly Leu Tyr Cys Asp Arg Gly Pro Glu 80 85 90 Asp Gly Gly Gly AlaGly Ile Cys Met Val Leu Glu Gly Asp Asn 95 100 105 Cys Val Phe Asp GlyMet Ile Tyr Arg Asn Gly Glu Thr Phe Gln 110 115 120 Pro Ser Cys Lys TyrGln Cys Thr Cys Arg Asp Gly Gln Ile Gly 125 130 135 Cys Leu Pro Arg CysAsn Leu Gly Leu Leu Leu Pro Gly Pro Asp 140 145 150 Cys Pro Phe Pro ArgLys Ile Glu Val Pro Gly Glu Cys Cys Glu 155 160 165 Lys Trp Val Cys AspPro Arg Asp Glu Val Leu Leu Gly Gly Phe 170 175 180 Ala Met Ala Ala TyrArg Gln Glu Ala Thr Leu Gly Ile Asp Val 185 190 195 Ser Asp Ser Ser AlaAsn Cys Ile Glu Gln Thr Thr Glu Trp Ser 200 205 210 Ala Cys Ser Lys SerCys Gly Met Gly Phe Ser Thr Arg Val Thr 215 220 225 Asn Arg Asn Gln GlnCys Glu Met Val Lys Gln Thr Arg Leu Cys 230 235 240 Met Met Arg Pro CysGlu Asn Glu Glu Pro Ser Asp Lys Lys Gly 245 250 255 Lys Lys Cys Ile GlnThr Lys Lys Ser Met Lys Ala Val Arg Phe 260 265 270 Glu Tyr Lys Asn CysThr Ser Val Gln Thr Tyr Lys Pro Arg Tyr 275 280 285 Cys Gly Leu Cys AsnAsp Gly Arg Cys Cys Thr Pro His Asn Thr 290 295 300 Lys Thr Ile Gln ValGlu Phe Arg Cys Pro Gln Gly Lys Phe Leu 305 310 315 Lys Lys Pro Met MetLeu Ile Asn Thr Cys Val Cys His Gly Asn 320 325 330 Cys Pro Gln Ser AsnAsn Ala Phe Phe Gln Pro Leu Asp Pro Met 335 340 345 Ser Ser Glu Ala LysIle 350 357 AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN 17 Met GlnSer Val Gln Ser Thr Ser Phe Cys Leu Arg Lys Gln Cys 5 10 15 Leu Cys LeuThr Phe Leu Leu Leu His Leu Leu Gly Gln Val Ala 20 25 30 Ala Thr Gln ArgCys Pro Pro Gln Cys Pro Gly Arg Cys Pro Ala 35 40 45 Thr Pro Pro Thr CysAla Pro Gly Val Arg Ala Val Leu Asp Gly 50 55 60 Cys Ser Cys Cys Leu ValCys Ala Arg Gln Arg Gly Glu Ser Cys 65 70 75 Ser Asp Leu Glu Pro Cys AspGlu Ser Ser Gly Leu Tyr Cys Asp 80 85 90 Arg Ser Ala Asp Pro Ser Asn GlnThr Gly Ile Cys Thr Ala Val 95 100 105 Glu Gly Asp Asn Cys Val Phe AspGly Val Ile Tyr Arg Ser Gly 110 115 120 Glu Lys Phe Gln Pro Ser Cys LysPhe Gln Cys Thr Cys Arg Asp 125 130 135 Gly Gln Ile Gly Cys Val Pro ArgCys Gln Leu Asp Val Leu Leu 140 145 150 Pro Glu Pro Asn Cys Pro Ala ProArg Lys Val Glu Val Pro Gly 155 160 165 Glu Cys Cys Glu Lys Trp Ile CysGly Pro Asp Glu Glu Asp Ser 170 175 180 Leu Gly Gly Leu Thr Leu Ala AlaTyr Arg Pro Glu Ala Thr Leu 185 190 195 Gly Val Glu Val Ser Asp Ser SerVal Asn Cys Ile Glu Gln Thr 200 205 210 Thr Glu Trp Thr Ala Cys Ser LysSer Cys Gly Met Gly Phe Ser 215 220 225 Thr Arg Val Thr Asn Arg Asn ArgGln Cys Glu Met Leu Lys Gln 230 235 240 Thr Arg Leu Cys Met Val Arg ProCys Glu Gln Glu Pro Glu Gln 245 250 255 Pro Thr Asp Lys Lys Gly Lys LysCys Leu Arg Thr Lys Lys Ser 260 265 270 Leu Lys Ala Ile His Leu Gln PheLys Asn Cys Thr Ser Leu His 275 280 285 Thr Tyr Lys Pro Arg Phe Cys GlyVal Cys Ser Asp Gly Arg Cys 290 295 300 Cys Thr Pro His Asn Thr Lys ThrIle Gln Ala Glu Phe Gln Cys 305 310 315 Ser Pro Gly Gln Ile Val Lys LysPro Val Met Val Ile Gly Thr 320 325 330 Cys Thr Cys His Thr Asn Cys ProLys Asn Asn Glu Ala Phe Leu 335 340 345 Gln Glu Leu Glu Leu Lys Thr ThrArg Gly Lys Met 350 355 184 AMINO ACIDS AMINO ACID <Unknown> LINEARPROTEIN 18 Met Lys Ser Val Leu Leu Leu Thr Thr Leu Leu Val Pro Ala His 510 15 Leu Val Ala Ala Trp Ser Asn Asn Tyr Ala Val Asp Cys Pro Gln 20 2530 His Cys Asp Ser Ser Glu Cys Lys Ser Ser Pro Arg Cys Lys Arg 35 40 45Thr Val Leu Asp Asp Cys Gly Cys Cys Arg Val Cys Ala Ala Gly 50 55 60 ArgGly Glu Thr Cys Tyr Arg Thr Val Ser Gly Met Asp Gly Met 65 70 75 Lys CysGly Pro Gly Leu Arg Cys Gln Pro Ser Asn Gly Glu Asp 80 85 90 Pro Phe GlyGlu Glu Phe Gly Ile Cys Lys Asp Cys Pro Tyr Gly 95 100 105 Thr Phe GlyMet Asp Cys Arg Glu Thr Cys Asn Cys Gln Ser Gly 110 115 120 Ile Cys AspArg Gly Thr Gly Lys Cys Leu Lys Phe Pro Phe Phe 125 130 135 Gln Tyr SerVal Thr Lys Ser Ser Asn Arg Phe Val Ser Leu Thr 140 145 150 Glu His AspMet Ala Ser Gly Asp Gly Asn Ile Val Arg Glu Glu 155 160 165 Val Val LysGlu Asn Ala Ala Gly Ser Pro Val Met Arg Lys Trp 170 175 180 Leu Asn ProArg 291 AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN 19 Met Gln ArgAla Arg Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu 5 10 15 Leu Val Leu LeuArg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser 20 25 30 Ser Gly Gly Leu GlyPro Val Val Arg Cys Glu Pro Cys Asp Ala 35 40 45 Arg Ala Leu Ala Gln CysAla Pro Pro Pro Ala Val Cys Ala Glu 50 55 60 Leu Val Arg Glu Pro Gly CysGly Cys Cys Leu Thr Cys Ala Leu 65 70 75 Ser Glu Gly Gln Pro Cys Gly IleTyr Thr Glu Arg Cys Gly Ser 80 85 90 Gly Leu Arg Cys Gln Pro Ser Pro AspGlu Ala Arg Pro Leu Gln 95 100 105 Ala Leu Leu Asp Gly Arg Gly Leu CysVal Asn Ala Ser Ala Val 110 115 120 Ser Arg Leu Arg Ala Tyr Leu Leu ProAla Pro Pro Ala Pro Gly 125 130 135 Asn Ala Ser Glu Ser Glu Glu Asp ArgSer Ala Gly Ser Val Glu 140 145 150 Ser Pro Ser Val Ser Ser Thr His ArgVal Ser Asp Pro Lys Phe 155 160 165 His Pro Leu His Ser Lys Ile Ile IleIle Lys Lys Gly His Ala 170 175 180 Lys Asp Ser Gln Arg Tyr Lys Val AspTyr Glu Ser Gln Ser Thr 185 190 195 Asp Thr Gln Asn Phe Ser Ser Glu SerLys Arg Glu Thr Glu Tyr 200 205 210 Gly Pro Cys Arg Arg Glu Met Glu AspThr Leu Asn His Leu Lys 215 220 225 Phe Leu Asn Val Leu Ser Pro Arg GlyVal His Ile Pro Asn Cys 230 235 240 Asp Lys Lys Gly Phe Tyr Lys Lys LysGln Cys Arg Pro Ser Lys 245 250 255 Gly Arg Lys Arg Gly Phe Cys Trp CysVal Asp Lys Tyr Gly Gln 260 265 270 Pro Leu Pro Gly Tyr Thr Thr Lys GlyLys Glu Asp Val His Cys 275 280 285 Tyr Ser Met Gln Ser Lys 290 206AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN 20 Met Leu Pro Pro AlaIle His Phe Tyr Leu Leu Pro Leu Ala Cys 5 10 15 Ile Leu Met Lys Ser CysLeu Ala Phe Lys Asn Asp Ala Thr Glu 20 25 30 Ile Leu Tyr Ser His Val ValLys Pro Val Pro Ala His Pro Ser 35 40 45 Ser Asn Ser Thr Leu Asn Gln AlaArg Asn Gly Gly Arg His Phe 50 55 60 Ser Asn Thr Gly Leu Asp Arg Asn ThrArg Val Gln Val Gly Cys 65 70 75 Arg Glu Leu Arg Ser Thr Lys Tyr Ile SerAsp Gly Gln Cys Thr 80 85 90 Ser Ile Ser Pro Leu Lys Glu Leu Val Cys AlaGly Glu Cys Leu 95 100 105 Pro Leu Pro Val Leu Pro Asn Trp Ile Gly GlyGly Tyr Gly Thr 110 115 120 Lys Tyr Trp Ser Arg Arg Ser Ser Gln Glu TrpArg Cys Val Asn 125 130 135 Asp Lys Thr Arg Thr Gln Arg Ile Gln Leu GlnCys Gln Asp Gly 140 145 150 Ser Thr Arg Thr Tyr Lys Ile Thr Val Val ThrAla Cys Lys Cys 155 160 165 Lys Arg Tyr Thr Arg Gln His Asn Glu Ser SerHis Asn Phe Glu 170 175 180 Ser Met Ser Pro Ala Lys Pro Val Gln His HisArg Glu Arg Lys 185 190 195 Arg Ala Ser Lys Ser Ser Lys His Ser Met Ser200 205

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide comprising amino acid −23 to amino acid 183 as set forth inSEQ ID NO:2; (b) a polynucleotide encoding the polypeptide comprisingamino acid 1 to amino acid 183 as set forth in SEQ ID NO:2 (c) apolynucleotide capable of hybridizing to and which is at least 70%identical to the polynucleotide of (a) or (b); and (d) a polynucleotidefragment of the polynucleotide of (a), (b) or (c).
 2. The polynucleotideof claim 1 wherein the polynucleotide is DNA.
 3. The polynucleotide ofclaim 2 which encodes the polypeptide comprising amino acid −28 to 183of SEQ ID NO:2.
 4. The polynucleotide of claim 2 which encodes thepolypeptide comprising amino acid 1 to 183 of SEQ ID NO:2.
 5. Anisolated polynucleotide comprising a member selected from the groupconsisting of: (a) a polynucleotide which encodes a mature polypeptideencoded by the DNA contained in ATCC Deposit No. (b) a polynucleotidewhich encodes a polypeptide expressed by the DNA contained in ATCCDeposit No. ______. (c) a polynucleotide capable of hybridizing to andwhich is at least 70% identical to the polynucleotide of (a) or (b); and(d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).6. The polynucleotide of claim 2 comprising the sequence as set forth inSEQ ID NO:1 from nucleotide 1 to nucleotide
 900. 7. The polynucleotideof claim 2 comprising the sequence as set forth in SEQ ID NO:1 fromnucleotide 117 to nucleotide
 735. 8. The polynucleotide of claim 2comprising the sequence as set forth in SEQ ID NO:1 from nucleotide 187to nucleotide
 735. 9. A vector containing the DNA of claim
 2. 10. A hostcell genetically engineered with the vector of claim
 11. 11. A processfor producing a polypeptide comprising: expressing from the host cell ofclaim 12 the polypeptide encoded by said DNA.
 12. A process forproducing cells capable of expressing a polypeptide comprisinggenetically engineering cells with the vector of claim
 11. 13. Apolypeptide comprising a member selected from the group consisting of(i) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2and fragments, analogs and derivatives thereof; and (ii) a polypeptideencoded by the cDNA of ATCC Deposit No. ______ and fragments, analogsand derivatives of said polypeptide.
 14. The polypeptide of claim 13comprising from amino acid 1 to amino acid 183 of SEQ ID NO:2.
 15. Acompound which mimics the activity of the polypeptide of claim
 13. 16. Acompound which antagonizes the activity of the polypeptide of claim 13.17. An antibody against the polypeptide of claim
 13. 18. A process foridentifying agonists and antagonists to the polypeptide of claim 13comprising: contacting a cell expressing on the surface thereof areceptor for the polypeptide, said receptor being associated with asecond component capable of providing a detectable signal in response tothe binding of a compound to said receptor, with a compound to bescreened under conditions to permit binding to the receptor; anddetermining whether the compound binds to and activates or inhibits thereceptor by detecting the presence or absence of a signal generated fromthe interaction of the compound with the receptor.
 19. A process fordiagnosing a disease or a susceptibility to a disease related to amutation in SCGF nucleic acid sequence comprising: determining amutation in the polynucleotide of claim
 1. 20. A diagnostic processcomprising: analyzing for the presence of the polypeptide of claim 13 ina sample derived from a host.